Partial purification and characterisation of transglutaminases from dicotyledonous plant tissue

Durose, LJ, 2001. Partial purification and characterisation of transglutaminases from dicotyledonous plant tissue. PhD, Nottingham Trent University.

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Abstract

Pisum sativum leaf tissue transglutaminase activity was found predominantly in the soluble fraction but approximately 10% of the activity was found in the microsomal membrane fraction. Microsomal transglutaminase could not be extracted from the membranes by high concentrations of salt or at pH 10, but was released from the membrane by the addition of 0.23% (w/v) sodium deoxycholate. Incubation of the washed microsomal membranes with trypsin released an active fragment of the enzyme. Discontinuous sucrose density gradient centrifugation of a mixed Pisum sativum microsomal membrane preparation showed that the microsomal transglutaminase activity was enriched in the tonoplast fraction. The soluble and microsomal forms of transglutaminase activity present in Pisum sativum leaf tissue showed similar biochemical characteristics including calcium ion requirement, response to thiol reactive reagents, substrate kinetics and inhibition by GTP when the activities were measured in the presence of sodiun deoxycholate. The microsomal transglutaminase activity could be detected in leaf tissue 10 days after germination and rose to a maximum 19 days after germination.

A calcium dependent transglutaminase from Vicia faba cotyledons was purified 500-fold by the calcium dependent binding and elution of the enzyme from both ion exchange and hydrophobic interaction chromatography resins. The molecular mass of the Vicia faba cotyledon transglutaminase was estimated to be 85 000 by SDS-PAGE and western blotting. The transglutaminase was activated at 10μM free calcium, was thiol dependent and had a pH optimum of 8.0. At sub-optimal calcium ion concentrations the activity was inhibited by GTP suggesting this enzyme could be regulated by GTP nucleotides. The Vicia faba cotyledon transglutaminase activity was detected 14 days after germination and reached a maximum 21 days after germination.

Item Type:	Thesis
Creators:	Durose, L.J.
Date:	2001
ISBN:	9781369316735
Identifiers:	Number Type PQ10183509 Other
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	30 Sep 2020 12:55
Last Modified:	13 Sep 2023 12:20
URI:	https://irep.ntu.ac.uk/id/eprint/41025

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